Our long-term goal is to understand the molecular mechanisms of BK channel activation. BK-type K+ channels are activated by voltage, intracellular Ca2+ and Mg2+. These channels are important in modulating muscle contraction, neural transmission and circadian pacemaker output. Recently, the voltage sensor, Ca2+ and Mg2+ binding sites in BK channels have been identified. However, the structural basis for the coupling between sensors and the activation gate, which are located in different structural domains, still remains elusive. A central question in this crucial step of BK channel gating is how these different structural domains interact with one another to mediate the coupling between the sensors and the activation gate. It has become apparent that a knowledge gap presented by this question is a critical barrier for understanding BK channel activation. Recent studies and our preliminary results lead to a general hypothesis for answering this question: interactions between the voltage sensor domain (VSD) and the cytosolic domain (CTD), and this interfacial alignment couples the ligand binding site on the CTD to the opening of the activation gate. We propose the following specific aims to examine three key aspects of this hypothesis. 1. To demonstrate that the electrostatic interactions affect the VSD-CTD alignment. 2. To demonstrate that the VSD-CTD alignment is coupled to the activation gate. 3. To show that the VSD-CTD interactions mediate coupling of Ca2+ binding to gate opening. We have developed innovative methods to measure VSD-CTD alignment and the coupling of this alignment to the activation gate. This study will identify amino acids and structural motifs important for BK channel activation and reveal the nature of the interactions among structural domains during this molecular process. A prevalent model proposed for ion channel activation by intracellular ligands is that ligand binding alters the conformation of the cytosolic domain, which pulls a peptide linker to open the activation gate. The results of our proposed study will show that in BK channels Mg2+ and Ca2+ may also activate the channel by pushing the voltage sensor via an electrostatic interaction involving the residues in different structural domains, which provides a novel mechanism of ligand dependent gating that may be shared by many other ion channels. BK channels are being pursued as therapeutic targets for neuronal ischemia, trauma and cognitive decline, and recent studies show that BK channels are associated with hypertension, schizophrenia, epilepsy and paroxysmal dyskinesia. The dissection of the molecular events during BK channel gating in this study will help identify specific targets for the development of therapeutics in addition to providing insights into the principles of ion channel gating.